Zeiss LSM 510 Confocor III Training Notes. Center for Cell Analysis & Modeling

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1 Zeiss LSM 510 Confocor III Training Notes Center for Cell Analysis & Modeling

2 Confocor 3 Start Up Go to System Module Turn on Main Switch, System/ PC, and Components Switches Do you need the arc lamp? Yes Turn on X Cite Lamp No Turn on remote System/PC and Components Switches located on the computer table. Log in to computer, CAM domain, and start AIM software

3 When prompted to login, press Ctrl + Alt + Del, be sure to log into the CAM domain. Start the Zeiss Aim Software In the LSM Switchboard, select Scan New Images and Start Expert Mode. DO NOT select Use Existing Images.

Creating/Accessing your database Select File, then New to create a new database or select Open for an existing database. Store all your data on the z partition (//cfs02.cam.uchc.

4 Creating/Accessing your database Select File, then New to create a new database or select Open for an existing database. Store all your data on the z partition (//cfs02.cam.uchc.edu/home/cam/your_ccam_account) that has been set up for your lab. DO NOT store data on the desktop or in My Documents. Please back up your data on your own devices. Note, there is a utility for Exporting your files to other file formats, however always be sure to save the raw data.

5 Database and LSM Files When you create a new database, the software will automatically create a new folder with the database name. In that folder, you will find the database file and your.lsm image files. These files are tiff files with Zeiss acquisition information stored in the header of the file. If you want to delete files from your database, do so using the Zeiss software rather than manually deleting the files directly from the windows folder. In the past, deleting the files manually has created problems with the database. You can open the files via the database with the free Zeiss LSM Image Browser, available from their web site, or with an ImageJ plugin, the Zeiss LSM Reader, Alternatively, the files themselves can be opened directly with Photoshop however you must use the Open As function and specify that it is a tiff file. Be aware that you can have two files with the same name in the database. The software will not warn you of file overwrite.

6 Lasers Go to the Acquire tab and select Laser to open the Laser Control dialog. Here you get access to the three lasers on the Confocor III: Argon (30mW) 458,477, 488, and 514 nm DPSS (Diode Pumped Solid-State) (1.0 mw) 561 nm HeNe (Helium-Neon)(5.0 mw) 633 nm Generally it is suggested the lasers need about a 30 minute warm up period but you can start using them straight away for non-quantitative imaging purposes.

Select the laser(s) that would provide the correct wavelength(s) for exciting your specimen. Argon This laser is fan cooled and has a specific start up and shut down procedure.

7 Select the laser(s) that would provide the correct wavelength(s) for exciting your specimen. Argon This laser is fan cooled and has a specific start up and shut down procedure. Select Standby, when Status indicates Ready, click On. Set the output to ~ 50%, that should correspond to a tube Current of ~ amps. The Argon laser offers the following lines: 458, 477, 488 and 514 nm. DPSS 561 and HeNe 633 The DPSS and helium-neon lasers are much weaker lasers and do not require fan cooling. Simply select the laser line required and press On to start the laser. Press Close, in the Laser Control dialog, once you have turned on the required laser(s).

Selecting Objectives It is easiest to define which objective(s) you will be using in the

position. Press Maintain>Objective to open the Objective Control dialog.

In the Change Objective dialog, find the objective you are using and double click your

8 Selecting Objectives It is easiest to define which objective(s) you will be using in the software, and then use the Microscope Control dialog to select the correct turret position. Press Maintain>Objective to open the Objective Control dialog. Click once to select a turret position, 1-6. In the Change Objective dialog, find the objective you are using and double click your selection. Repeat this procedure for any other objectives you may have. Close both dialogs once you have made your selections and return to the Acquire tab.

Select Microscope Tab Access microscope by selecting VIS Transmitted Light Microscope Control While on the Acquire tab, select VIS for directing the light to the microscope, and then select Micro to

9 Select Microscope Tab Access microscope by selecting VIS Transmitted Light Microscope Control While on the Acquire tab, select VIS for directing the light to the microscope, and then select Micro to access the Microscope Control dialog. From this dialog you can select your objective, and view your sample via transmitted light or fluorescent light. Press the Objective icon, and select your objective from the pull down menu. Make sure the Reflector cube is in the None position so the transmitted light does not have to pass through a colored filter. Press the Transmitted Light icon to access the Transmitted Light dialog. Press On and move the slider to adjust the intensity of the bulb. BE CAREFUL WITH THE INTENSITY SLIDER. You really shouldn t need to go above 1%. Deselect On to turn the bulb off and press Close to close the Transmitted Light box.

Microscope Control (cont.) Fluorescent Light Control You should still be in the VIS position on the Acquire tab, and have the Microscope Control dialog open.

10 Microscope Control (cont.) Fluorescent Light Control You should still be in the VIS position on the Acquire tab, and have the Microscope Control dialog open. Press Reflector to select the appropriate fluorescent filter. Select Reflected Light icon to open the shutter, this will let fluorescent light reach the objectives for finding your sample. Be sure to center your sample in the middle of the field. The scan area is very small in comparison to the field of view from the microscope. When you are finished viewing your sample; deselect the Reflected Light icon to close the shutter.

11 Using the Confocal Laser Scanning Mode With the Acquire tab selected, select the LSM tab to change to confocal mode. Press Config to open the Configuration Control dialog. Here you will choose between Multi Track and Single Track configurations and make your laser, emission filter and detector selections. All configurations can be stored for future use. Single Track Collects one probe or multiple probes simultaneously. Multi Track Collects multiple probes sequentially; by line or by frame. Does not prevent bleed through between channels. Avoids bleed through between channels Better for sample because of reduced exposure to laser. Exposes sample to increased laser light due to multiple scans. Faster acquisition Slower acquisition

12 Single Track Emission Filter Select Channel Mode and Single Track. Follow the excitation/emission pathway in the image to define your configuration. Press Excitation to select laser line and % transmission. Press and select the Excitation dichroic; this selection depends on the laser lines being used to excite the sample. Select 1 0 and 2 0 dichroics; selection is based upon wavelengths you want to capture. Dichroics reflect short wavelengths and pass long wavelengths. Activate detectors; Ch1, Ch2 and/or Ch3 and select psuedocolor for display. ChD is for transmitted light which is not confocal. 1 0 and 2 0 dichroics Three Photomultiplier tubes Transmitted light detector Excitation Dichroic

Multi Track Emission Filter Select Channel Mode and Multi Track. Follow the excitation/emission pathway in the image to define your configuration.

13 Multi Track Emission Filter Select Channel Mode and Multi Track. Follow the excitation/emission pathway in the image to define your configuration. Press Add Track for each fluorescent probe or combination of probes. Activate the track by selecting the check box. Press Excitation to select laser line and % transmission. Press and select the Excitation dichroic; this selection depends on the laser lines being used to excite the sample. Select 1 0 and 2 0 dichroics; selection is based upon wavelengths you want to capture. Dichroics reflect short wavelengths and pass long wavelengths. Activate detectors; Ch1, Ch2 and/or Ch3 and select psuedocolor for display. ChD is for transmitted light which is not confocal. The individual tracks can be saved separately or the entire configuration can be saved for later use. 1 0 and 2 0 dichroics Excitation Dichroic Three Photomultiplier tubes Transmitted light detector

14 Emission and Dichroic Filter Selections Channel 1 Channel 2 Tertiary Dichroic Primary Dichroic Secondary Dichroic Channel 3

15 Photomultiplier Tubes (PMTs) and Avalanche Photodiodes (APDs) PMTs extremely sensitive detectors of ultraviolet, visible and near-infrared light. Detectors can multiply the current produced by the light by as much as 100 million times. Characteristics: high gain, low noise, high response, large area of collection. Limited sensitivity in red and near infrared regions. APDs Semiconductor device used as an alternative to PMTs. Can measure light at lower levels; used for single photon counting. Improved sensitivity in red and near infrared regions, nm.

16 Dichroics to APDs Emission Filters APD 2 Protects APDs when not in use APD 1

If you have multiple channels, start with the longest wavelength and set the airy unit to 1.

17 Scan Control Channels Tab Under the Acquire tab, select the Scan button to get to the Scan Control Dialogue, press the Channels tab. Set the pinhole for each channel. Start at 1 airy unit for the best theoretical resolution. If you have multiple channels, start with the longest wavelength and set the airy unit to 1. Make sure the other channls have the same Optical Slice, adjust the pinhole if necessary. Adjust the Detector Gain, maximum sensitivity, and the Amplifier Offset, minimum intensity, for your image settings. 1 Airy Unit

) Adjust the Detector Gain and Amplifier Offset to remove red and blue pixels.

18 Scan Control Channels Tab (cont.) Select the Palette tab, on the image display window, choose Range Indicator, then close the dialog. Depending on the settings, your image will display Blue pixels for minimum intensity and Red pixels for maximum intensity. ( for 8 bit images and for 12 bit images.) Adjust the Detector Gain and Amplifier Offset to remove red and blue pixels. The minimum pixel intensity should be ~ gray levels above 0 and the maximum pixel intensity should be ~10-20 gray levels below 255. If you have multiple channels, use Split x,y to view the channels separately as well as the over-lay image. In the MultiTrack configuration, you can deselect a channel in the Configuration Control to scan them one at a time when adjusting the settings. Once you have the settings for each channel, activate all the channels and perform a single scan generating the over-lay image. Use the range indicator to aid with image settings. Split x,y Palette>Range Indicator

19 Scan Control Mode Tab Objective change lens from this window or from the microscope dialog (recommended) Frame Size size of image, defaults to 512x512, ranges from 128 to 2048 pixels in x and y. Line Step sampling rate of each frame. Recommended to leave at 1 for best image quality. Scan Speed slower scan allows for longer pixel dwell time which will result in better image quality but result in increased chance of photobleaching. Data Depth select between 8 bit (256 gray levels) and 12 bit (4095 gray levels) Use 12 bit for quantitative, data analysis. Scan Direction use single direction for fixed cells, bidirectional for faster, live cell acquisition. Scan Average improves signal to noise ratio Mode Line or Frame Method Mean or sum Number of lines or frames to average by. Decreased scan speed and line averaging result in increased laser exposure. Frame averaging reduced photobleaching but does not produce as clean an image as the other methods.

20 Scan Control Mode Tab(cont.) Zoom Use the Crop tool, on the image display window, to implement the zoom function, and/or rotation function. Rest image to zoom 1 when you are finished. This will center the laser to optimal conditions. The final magnification is zoom factor x magnification of the objective.

21 Nyquist Sampling In order to preserve the spatial resolution in an image, the sampling rate should be twice that of the maximum frequency. Adjust the optical zoom in order to achieve the optimal x,y pixel size. Objective Numerical Aperture Immersion Coverslip Thickness (mm) Working Distance (mm) Z Resolution (μm) (1 Airy Unit) *Optimal Z-Step (μm) **XY Resolution (μm) *Optimal XY pixel size ***Optimal Zoom 40x C- Apochromat 1.2 Water x Plan- Apochromat.95 Air x Plan- Apochromat 1.4 Oil x Plan- Apochromat 1.4 Oil x Plan- Neofluar.3 Air x Plan- Neofluar.5 Air x Plan- Neofluar 1.25 Oil x Fluar.5 Air x Fluar 1.3 Oil

Scan Control Z-Settings Z-Stack Stack Z size total size of stack Focus current focus position Z-sectioning You can define your stack by the number of slices, the step interval and the current slice

22 Scan Control Z-Settings Z-Stack Stack Z size total size of stack Focus current focus position Z-sectioning You can define your stack by the number of slices, the step interval and the current slice position you are at. Collect the stack by either Keeping the Interval (number of slices will be modified) or Keeping the Slice (the interval will be modified). Mark First/Last focus to the bottom of your sample and select Mark First, focus to the top of your sample, Mark Last. Interval spacing at which you will acquire the slices. This should be based on the optimal Z-slice, which is equal to one half the z-resolution. Press the Z Slice button for this to be calculated automatically. Note scan buttons are different under Z settings.

23 Scan Control Z-Stack - Z-Settings (cont.) Move to First, Mid, Last automatically move to position once the first and last positions have been set. Refr. Corr. corrects for mismatch between immersion oil and mounting medium. Auto Z Corr. compensates for signal loss in the volume by increasing laser power and detector gain. Move to First, Mid, Last automatically move to position once the first and last positions have been set. Refr. Corr. corrects for mismatch between immersion oil and mounting medium. Auto Z Corr. compensates for signal loss in the volume by increasing laser power and detector gain. Note scan buttons are different under Z settings.

24 Scan Control Z-Stack - Z-Settings (cont.) Note the scan buttons change under the Z Settings tab Start starts z-stack Stop stops scans XY Scan single scan XY cont continuous scan Line Sel line scan Note scan buttons are different under Z settings.

25 3D View Under the 3D View tab, there are primitive functions that allow you to view your 3D data set. Compile 3D stack for display

Shutting down the Lasers Shutdown Procedure (Last User) Argon Laser Lower power, select Standby, select Off. Wait until cooling cycle ends, ~ 3 minutes, Status will be reported as Connected.

26 Shutting down the Lasers Shutdown Procedure (Last User) Argon Laser Lower power, select Standby, select Off. Wait until cooling cycle ends, ~ 3 minutes, Status will be reported as Connected. HeNe 1 and HeNe 2 Select Off for each laser Shutdown Procedure (Between users; verify next user is coming. If in doubt, shut it down.) Argon Laser Lower power, select Standby. HeNe 1 and HeNe 2 Select Off for each laser Log off from computer, DO NOT shut the system off when the lasers are still on as you will damage the lasers.

27 Check the CC3 sign up prior to shutting down the system. Confocor 3 Shut Down Is someone signed up after you? Yes Did you verify they are coming? Yes Leave the system in a stand by mode and log off. No No SYSTEM SHUT DOWN: Turn off lasers, exit AIM software Shut down computer Turn off X Cite bulb Turn off remote System/PC and Components Switches. Go to System Module Turn off System/ PC, Components Switches, and Main Switch.

LSM 510 Meta Training Notes

LSM 510 Meta Training Notes Turning on the system Turn on X-Cite power supply. This supplies light for epifluorescence for viewing your samples through the microscope. Turn on the remote control switch.